Public health concems have emerged regarding use of smallpox as a bioterrorist weapon since most Americans are no longer immune. Poxviruses subvert the complement system via the expression of regulatory proteins. In variola, vaccinia and ectromelia, the proteins are called SPICE (for smallpox inhibitor of complement enzymes), VCP (vaccinia virus complement control protein) and EMICE (an uncharactedzed analog in ectromelia that we have labeled "ectromelia inhibitor of complement enzymes"). These secreted virulence factors down-regulate complement activation by mimicking the functional repertoire of a family of host proteins called the Regulators of Complement Activation (RCA). The viral proteins are also structurally related to their host counterparts. Specific Aims: 1. To characterize the complement inhibitory profile of SPICE compared to its human counterparts. We will identify the principal complement-evading activity of SPICE and this will become a target for neutralization. These assessments will take place with the native soluble protein as well as after it attaches to cells via either its heparin-binding site(s) or by addition of an anchor. 2. To determine the complement regulatory sites of SPICE. These experiments will take advantage of the functional profiles (defined in Aim 1) and the sequences of active sites of RCA proteins that are homologous to corresponding regions of SPICE, VCP, and EMICE. These two sets of data provide a logical strategy for a mutational analysis to locate the active sites. 3. To characterize the complement regulatory activity of EMICE. This mousepox protein has not been evaluated for its complement inhibitory (virulence) activity. It is about 90% identical to SPICE and VCP. We will first characterize its regulatory activity for human and mouse complement. Second, we will assess its role in vivo as a virulence factor by infecting sensitive and resistant mouse strains with the ectromelia virus deleted of its complement regulator. The proposed experiments should provide novel information relative to the pathogenesis of poxvirus infections of man and mouse. Additionally, these results will serve as a guide to produce a less toxic small pox vaccine and to identify a viral target for mAb treatment of variola infection.